HOW DIDEMNINS INHIBIT PROTEIN BIOSYNTHESIS

地德米宁如何抑制蛋白质生物合成

• 批准号: 2008379
• 负责人: PETER L. TOOGOOD
• 金额: $ 10.05万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-12-17 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2008379
• 关键词: Tunicata; animal extract; antineoplastics; antiviral agents; cell free system; chemical structure function; crosslink; cyclic peptides; drug design /synthesis /production; growth inhibitors; immunomodulators; inhibitor /antagonist; protein biosynthesis; radiotracer

The marine natural product didemnin B displays potent antiviral, antitumor and immunomodulatory effects in cultured cells and in vivo. It has entered phase-II clinical trails as an antitumor agent. The reasons why didemnin B displays such potent biological activity are not understood, however didemnin B has been shown to inhibit the biosynthesis of DNA, RNA and proteins in whole cells. We have demonstrated the inhibition of in vitro protein synthesis by didemnin B and didemnin A, using a Cell-free translation system derived from rabbit reticulocytes, and have shown that didemnin B is equally effective as an inhibitor of protein synthesis in wheat germ. Furthermore, this effect is attenuated by modification of didemnin B as its bis(O-acetyl)derivatives. In our preliminary results, we present data that support he hypothesis that didemnin B inhibits protein synthesis specifically at eh elongation stage. Further studies are proposed to elucidate the detailed mechanism of inhibition of protein synthesis by didemnin B in vitro. Specifically, experiments are described that will define the precise step of elongation that is affected (amino acid delivery, peptide bond formation, or translocation), based on the effect of didemnin B upon protein synthesis partial reactions in rabbit reticulocyte or yeast lysates. The cytosolic molecular target of didemnin B will be identified using radiolabeled didemnin derivatives, or by cross-linking with a didemnin ligand. The design and synthesis of a new cross-linking agent is described. The effect of didemnin B upon regulated via phosphorylation of several translation factors and ribosomal proteins. The phosphoproteins will be labeled with 32P and resolved by tow-dimensional gel electrophoresis. To identify which structural features of didemnin B are important for its inhibition of protein synthesis, didemnin analogs will be synthesized and tested as inhibitors of in vitro protein synthesis. The structure- function profile for the didemnins that is obtained from these studies will be used in the design of new potential inhibitors of translation in ukaryotes. It is hypothesized that other potent inhibitors of protein synthesis besides the didemnins will also be effective antitumor agents, and this hypothesis will be tested by the synthesis and evaluation of didemnin mimics and limited screening of related natural products.

海洋天然产物 didemnin B 显示出有效的抗病毒作用， 在培养细胞和体内具有抗肿瘤和免疫调节作用。 作为抗肿瘤药物，它已进入 II 期临床试验。 这 地德明宁 B 表现出如此强大的生物活性的原因并非如此 理解，然而，didemnin B 已被证明会抑制生物合成 全细胞中的 DNA、RNA 和蛋白质。 我们已经证明了 地德明宁 B 和地德明宁 A 抑制体外蛋白质合成， 使用源自兔网织红细胞的无细胞翻译系统， 并表明，didemnin B 作为抑制剂同样有效 小麦胚芽中的蛋白质合成。 此外，这种影响会减弱 通过将didemnin B修饰为其双(O-乙酰基)衍生物。 在我们的 初步结果，我们提供的数据支持他的假设 didemnin B 在 eh 延伸时特异性抑制蛋白质合成 阶段。 建议进一步研究以阐明其详细机制 体外 Didemnin B 抑制蛋白质合成。 具体来说， 描述的实验将定义精确的伸长步骤 受影响的部分（氨基酸输送、肽键形成或 易位），基于 didemnin B 对蛋白质合成的影响 兔网织红细胞或酵母裂解物中的部分反应。 细胞质的 didemnin B 的分子靶标将使用放射性标记来鉴定 地德明宁衍生物，或通过与地德明宁配体交联。 这 描述了一种新型交联剂的设计和合成。 这 地德明宁 B 通过几种磷酸化调节的作用 翻译因子和核糖体蛋白。 磷蛋白将是 用 32P 标记并通过二维凝胶电泳解析。 确定 didemnin B 的哪些结构特征对其重要 抑制蛋白质合成，将合成didemnin类似物 经测试作为体外蛋白质合成的抑制剂。 结构- 从这些研究中获得的 didemnin 的功能概况 将用于设计新的潜在翻译抑制剂 核生物。 据推测，其他有效的蛋白质抑制剂 除didemnins之外的合成也将是有效的抗肿瘤剂， 这个假设将通过综合和评估来检验 didemnin 模拟物和相关天然产物的有限筛选。